Chemotherapy using cytotoxic drugs is presently the most commonly used weapon in the treatment of advanced cancer. However, the therapeutic efficacy of cancer chemotherapeutic drugs is often limited by factors such as tumor heterogeneity, drug resistance and host toxicity. Recent advances in molecular medicine have provided new opportunities to improve cancer chemotherapeutics through the development of novel, more selective anti-cancer strategies. This proposal builds on progress made during the last project period in developing a novel, cytochrome P450 prodrug activation-based gene therapy, whose therapeutic potential for cancer treatment derives, in part, from the striking cytotoxicity enhancement that is associated with intratumoral, as compared to hepatic P450-catalyzed prodrug activation. This approach is unique among current prodrug activation strategies, insofar as it utilizes mammalian genes in combination with clinically useful anti-cancer prodrugs, rather than novel prodrugs whose ultimate therapeutic efficacy is uncertain. The present application aims to test a series of hypotheses developed to further improve the responsiveness of tumor cells to the widely used, liver P450-activated oxazaphosphorine anti-cancer prodrug cyclophosphamide, both in the context of conventional chemotherapy and P450-based gene therapy for cancer. The major aims of this proposal are: 1) to enhance the responsiveness of tumor cells to oxazaphosphorine therapy by targeting aldehyde dehydrogenase enzymes, which serve as major cellular determinants of cyclophosphamide resistance; 2) to investigate the role of a tumor endothelial cell-directed bystander effect in the action of P450 gene therapy, and to determine whether this effect can be enhanced by metronomic scheduling of cyclophosphamide, either alone or in combination with anti-angiogenic agents; and 3) to enhance tumor cell bystander cytotoxicity, and hence the overall efficacy of P450 gene therapy, by introduction of caspase inhibitors that delay, but do not block, the death of 'P450 factory' tumor cells. Together, these studies will establish a rational basis for increasing the activity of cyclophosphamide and other P450 prodrugs in a manner that moderates toxic host responses and improves therapeutic effects, and will thereby advance the development and implementation of P450 prodrug-based therapies for cancer treatment [unreadable] [unreadable]